new rp-hplc method for the determination of afatinib ...

Indo American Journal of Pharmaceutical Research, 2015

ISSN NO: 2231-6876

NEW RP-HPLC METHOD FOR THE DETERMINATION OF AFATINIB DIMALEATE IN BULK AND PHARMACEUTICAL DOSAGE FORMS Ravikumar.Vejendla*1, CVS Subramanyam2, G Veerabhadram3. 1

Sri Indu Institute of Pharmacy, Sheriguda, Ibrahimpatnam, Hyderabad. Osmania University, Department of Chemistry, Hyderabad. 3 Gokaraju Rangaraju College of Pharmacy, Hyderabad. 2

ARTICLE INFO Article history Received 08/04/2015 Available online 30/05/2015 Keywords Afatinib-Dimaleate, Gilotrif, Liquid Chromatography, Method Development And Validation.

ABSTRACT A novel stability indicating liquid chromatographic assay method was developed and validated as per ICH guidelines for the quantitative estimation of Afatinib in tablet formulation. An gradient reverse phase LC-method was developed using X-Terra RP-8, 250 x 4.6mm, 5μm column and a mobile phase were aqueous Potassium dihydrogen orthophosphate buffer adjusted pH at 3.0 with o-phosphoric acid (mobile phase solvent-A) and Acetonitrile:Methanol(70:30v/v) (mobile phase solvent-B) in a gradient mode. The detector set at 258nm with flow rate of 1.0mL min-1. The method is linear between 0.12 to 0.36 mg/mL with a good linear relationship (r2=0.998) was observed and the limit of detection (LOD) is 0.06 μg/ mL 0.02% and limit of quantification (LOQ) is 0.06 mg/mL 0.06%. The Accuracy of the method was found to be in the range of 99.70% to 100.26%. The mean Inter and Intraday Precision has Relative Standard deviation (%RSD) were less than 0.147. Robustness was done by change in column, variation in change of flow rate of +/- 0.2 mL/min. the values were found to be within the limits. The Proposed method was found to be Linear, precise and accurate for the quantitative estimation of Afatinib in tablet formulations and can be used for commercial purposes.

Copy right © 2015 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

www.iajpr.com

Page

Please cite this article in press as Ravikumar.Vejendla et al. New RP-HPLC Method For The Determination of Afatinib Dimaleate in Bulk And Pharmaceutical Dosage Forms. Indo American Journal of Pharm Research.2015:5(05).

2098

Corresponding author Ravikumar.Vejendla Sri Indu Institute of Pharmacy, Sheriguda, Ibrahimpatnam, Hyderabad.

Vol 5, Issue 05, 2015.

Ravikumar.Vejendla et al.

ISSN NO: 2231-6876

INTRODUCTION Monoclonal antibodies targeting vascular endothelial growth factor (VEGF) or the epidermal growth factor receptor (EGFR) represent well-established treatment options for colorectal cancer. The VEGF antibody bevacizumab enhances the efficacy of oxaliplatin-based and irinotecan-based chemotherapy1-3, presumably by normalization of the tumour vasculature 4-7. EGFR antibodies, such as cetuximab and panitumumab, may act on the tumour cells directly, inhibiting cellular growth, differentiation and proliferation, and inducing antibody dependent cell-mediated cytotoxicity8,9. Monoclonal antibodies against the EGFR have demonstrated activity as mono-therapy in pretreated patients10,11. Cetuximab in combination with chemotherapy also significantly prolongs progression-free survival (PFS) in the first-line treatment of patients with metastatic colorectal cancer compared with chemotherapy alone 12. Simultaneous targeting of tumour cell receptors may also offer the potential for synthetic lethality of therapeutic agents that have little activity as mono-therapy13-16, and cross-talk of pathways may involve mechanisms that can be used to overcome resistance 17.Afatinib is a novel, potent and irreversible inhibitor of both the epidermal growth factor receptor (EGFR) / human epidermal growth factor receptor (HER)1 and HER2 kinases. Preclinical studies demonstrate that afatinib has effective anti-tumour activity in a variety of human xenograft models. Phase I studies have shown that afatinib is well tolerated across a range of different dosing schedules, the maximum tolerated dose (MTD) initially being defined as 70 mg once daily for non-continuous dosing of afatinib. Several phase II trials yielded promising results in non-small cell lung cancer (NSCLC) patients with EGFR mutations. Phase III trials in NSCLC and breast cancer are currently ongoing18. Chemistry and Mechanism of action: Afatinib is presented as the dimaleate salt, with the chemical name N-[4-[(3-Chloro-4-fluorophenyl)amino]-7-[[(3S)tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2-butenamide Its structural formula is given in Fig.1. Afatinib dimaleate is a white to brownish yellow powder, water soluble and hygroscopic, with an empirical formula of C32H33ClFN5O11, and a molecular weight of 718.1 g/mol.19 Mechanisam of action for Afatinib is like lapatinib and neratinib, afatinib is a tyrosine kinase inhibitor (TKI) that also irreversibly inhibits human epidermal growth factor receptor 2 (Her2) and epidermal growth factor receptor (EGFR) kinases. Afatinib is not only active against EGFR mutations targeted by first generation TKIs like erlotinib orgefitinib, but also against those not sensitive to these standard therapies. Because of its additional activity against Her2, it is being investigated for breast cancer as well as other EGFR and Her2 driven cancers20.

Figrue-1: Chemical Structure of Afatinib dimaleate. Table 1.1: Physico-Chemical Properties: State/Color/Form Solubility

Bioavailability Half life Excretion CAS number

White to brownish yellow powder Very poorly soluble in dimethyslulfoxide (DMSO); ethanol and soluble in water at 100mg/mL; buffers or other additives may increase the aqueous solubility 72% 37 hours Faces (85%), urine (4%) 439081-18-2

Proprietary Name Gilotrif ®

Company Boehringer Ingelheim

Pharmaceutical Formulations It is a tablet for oral administration available in 40 mg, 30 mg, or 20 mg of afatinib (equivalent to 59.12 mg, 44.34 mg, or 29.56 mg afatinib dimaleate, respectively).

Page

S.No 1

2099

Table 1.2: Pharmaceutical Formulations of Afatinib dimaleate.

www.iajpr.com

Vol 5, Issue 05, 2015.


ISSN NO: 2231-6876

Motivation for the method development: The primary purpose of this research project was to develop and to validate a simple, precise and accurate HPLC method for determination of Afatinib dimaleate in the Bulk and in finished product. Clearly, it is highly important to accurately measure its concentration alone. A high speed method was sought to measure the concentration of this compound within a short span of time. This is beneficial in any pharmaceutical analysis/clinical environment where the concentration of Afatinib dimaleate is needed to understand any patient issues along with the pharmaceutical industry to prepare the multiple steps that may be needed to prepare the raw material for production. The high speed method will eliminate/reduce any waste or costs that are required with the preparation of the raw material. There are no analytical methods have been reported for the estimation of Afatinib dimaleate in bulk and in pharmaceutical formulations at the time of commencement of research work. The present HPLC method deals with new simple, accurate and reliable estimation of Afatinib dimaleate in tablet dosage form which have been not reported earlier. The review articles had been reported on therapeutic drug monitoring of anticancer drugs part two – targeted therapies21, Gefitinib and afatinib treatment in an advanced nonsmall cell lung cancer (NSCLC) patient undergoing hemodialysis22 and pharmacokinetics and metabolism after oral administration to healthy male volunteers mainly on the determination of Afatinib dimaleate23 in plasma, blood samples and biological fluids including tissue homogenates. The determination of Afatinib dimaleate in a Raw Material sample is yet to be found. In addition, stability-indicating methods have been able to be found for the Afatinib dimaleate in pharmaceutical dosage forms. Complete validation parameters were not able to be found in any of the methods reported in the past. Studying the stability of a drug and being able to monitor degradation products aids in the clinical treatments/early product development and shelf life for the drug. Hence, by considering all these factors, the author has made some humble attempts, hoping to fill this gap, and succeeded in developing analytical methods using HPLC methods. Table 1.3: Structural Features of Afatinib dimaleate: Official name Afatinib dimaleate

Chemical Name(s) N-[4-[(3-Chloro-4fluorophenyl)amino] -7-[[(3S)-tetrahydro-3-furanyl]oxy] -6-quinazolinyl]-4(dimethylamino)2-butenamide

Structure 1. 2. 3.

Functional groups Aromati primary amine. Carbanyl group. Pterdine nucleus

HPLC Estimation of Afatinib dimaleate: This part of the thesis reports sensitive and precise HPLC method for the determination of drug in bulk and in pharmaceutical formulations. Afatinib dimaleate was found to be completely soluble in acetonitrile and also, Afatinib dimaleate has UV absorption maxima at 258 nm. Hence conventional reverse phase HPLC has been selected for its estimation in pharmaceutical dosage forms. Experimental: Quantitative HPLC was performed on Agilent liquid Chromatography, with PDA detector equipped with automatic injector with injection volume 20 µl, and 2693 pump. X-Terra RP-8, 250mmx4.6mm, 5µ was used. The HPLC system was equipped with LC solution Software. Preparation of Standard drug solution: A standard stock solution of the drug was prepared by dissolving 150.2mg of Afatinib working standard into a 50ml volumetric flask, dissolve in 30 ml of diluent, sonicated for about 15 min and then made up to 50 ml with and dilute. Take 5ml into 50ml volumetric flask dilute with diluent to get approximately 0.3004mg/mL. Preparation of Sample solution: Weigh and transfer 300.4mg of Gilotrif tablet (by crushing 20 tablets in mortar and passel) powder into a 50ml volumetric flask; dissolve it with 30ml of diluent. The mixture was allowed to stand for 1 hr with intermittent sonication for complete solubility of the drug, filter through a 0.45 μm membrane filter, followed by addition of diluent up 50 ml. Transfer 5ml to 50ml volumetric flask dilute with diluent to get the stock solution of 0.6008mg/ml. The resultant solution was used as working solution.

www.iajpr.com

Page

Analytical Methodology: Preparation of Mobile phase: The contents of the mobile phase were aqueous Potassium dihydrogen orthophosphate buffer adjusted pH at 3.0 with orthophosphoric acid (mobile phase solvent-A) and acetonitrile : methanol in the ratio of 70:30v/v (mobile phase solvent-B) in a gradient mode of separation was used to resolute the pemetrexed disodium. They were filtered before use through a 0.45 μm membrane filter and degassed by sonication. The gradient program has been shown in Table.

2100

Reagents Used: Acetonitrile, methanol HPLC grade (Qualigens) and orho-Phosphoric acid (Rankem)

Vol 5, Issue 05, 2015.


ISSN NO: 2231-6876

Table No 1.4 Gradient flow usage of Mobile phase. Time in minutes 0 15 16 18

Mobile phase solvent-A 90 20 90 90

Mobile phase Solvent-B 10 80 10 10

Chromatographic Conditions: Column : X-Terra RP-8, (250mmx4.6mm, 5µ). Mobile phase : Solvent-A is potassium dihydrogen orthophosphate buffer system adjusted pH-3.0 with o-phosphoric acid and Solvent-B is Acetonitrile:methanol (70:30v/v Diluent : Water: Acetonitrile (30:70 v/v) Flow rate 1.0 ml/min. Run time 20 min Temperature Ambient. Injection volume : 10 μl Detection wavelength 258 nm Retention time : 10.558 min Recommended Procedure: After systematic and detailed study of the various parameters involved as described under results and discussion in this chapter, the following procedure was recommended for the determination of Afatinib dimaleate in bulk and pharmaceutical formulations. Working Standard Solution: 1ml of the Standard stock solution was taken in 10 ml volumetric flask and thereafter made up to 10 ml with mobile phase to get a concentration of 300.4µg/ml. Procedure: Initially the mobile phase was pumped for about 30 min, to saturate the column thereby to set the baseline corrected. Then 10μl of Afatinib dimaleate standard and sample solution were injected separately. A quantitative determination of the active ingredient was made by compare the peak area of a sample injection to the corresponding peak area of a standard injection. The amount of Afatinib dimaleate present in the sample was calculated through the standard calibration curve. RESULTS AND DISCUSSIONS Method Validation: The following parameters were used to validate the method for the estimation of Afatinib dimaleate in bulk sample and in tablet dosage form.

Page

2101

System Suitability: The system suitability tests were carried out on freshly prepared standard stock solution of Afatinib dimaleate. The system was suitable for use, the tailing factor was 1.228, peak area was 7242599 and USP theoretical plates were found to be significantly high around 30569.574. This was given in fig. 1.

www.iajpr.com

Vol 5, Issue 05, 2015.


ISSN NO: 2231-6876

Fig-1: Typical chromatogram for system suitability for Afatinib dimaleate. Specificity: The effect of wide range of intermediates and other precursors, generally used in pharmaceutical formulations of Afatinib dimaleate were investigated under optimized chromatographic conditions. Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include impurities, de-gradients, matrix, etc. The common excipients present in the pharmaceutical dosage form did not interfere with the elution or quantification of the method. Each 20 mg of Gilotrif tablet is equivalent to 29.56 mg afatinib dimaleate. Acceptance criteria for specificity, RSD should be less than 2%. The specificity chromatograms are shown in the Figure 1.1, 1.2 and 1.3. The assay of Afatinib dimaleate in tablet dosage form was found to be 99.38%. From the recovery studies it was found that about 103.13% on average of Afatinib dimaleate was recovered which indicates high accuracy of the method.

Fig-1.2: Typical Chromatogram of Afatinib dimaleate working standard solution.

www.iajpr.com

Page

2102

Fig 1.1: Typical Blank Chromatogram of Afatinib dimaleate Assay method.

Vol 5, Issue 05, 2015.


ISSN NO: 2231-6876

Fig-1.3: Typical Chromatogram of Afatinib dimaleate (Gilotrif) Working sample solution. Precision: The precision of the method was ascertained separately from the peak area obtained by actual determination of 6 replicas of a fixed amount of drug and formulation. The HPLC systems was set up the described Chromatographic conditions, mentioned as above and follow the system to equilibrate, and then injected the 0.3mg/ml concentration of afatinib dimaleate standard 6 times and recorded the response (peak area) given in fig. 2.1. The proposed method was extended to the pharmaceutical dosage forms by injecting the 0.6mg/ml of afatinib dimaleate sample (Gilotrif) 6times recorded the response (peak area) in Fig.2.2. The percent relative standard deviation and percent range of error (at 0.05 and 0.01 confidence limits) were calculated and presented in Table 2.1 for standard and 2.2 for sample. .

Fig 2.1: Chromatogram to illustrate Precision of Standard drug. Table 2.1: Precision of Standard drug with statistics: Sample name Afatinib / Precision Afatinib / Precision Afatinib / Precision Afatinib / Precision Afatinib / Precision Afatinib / Precision

Retention time 10.556 10.561 10.561 10.552 10.558 10.559 10.558 0.033 0.003

Area 7235770 7221253 7235122 7225670 7252082 7233728 7233937 0.147 10614

Page

2103

S.No. Title 1 230814_5.1cd 2 230814_6.1cd 3 230814_7.1cd 4 230814_8.1cd 5 230814_9.1cd 6 230814_10.1cd Average % RSD Std. Deviation

Fig 2.2: Chromatogram to illustrate Precision of afatinib dimaleate sample (Gilotrif).

www.iajpr.com

Vol 5, Issue 05, 2015.


ISSN NO: 2231-6876

Table 2.2: Precision study of Sample (Gilotrif ® 20 mg tablet) with statistics: S.No. Title 1 230814_5.11cd 2 230814_6.12cd 3 230814_7.13cd 4 230814_8.14cd 5 230814_9.15cd 6 230814_10.16cd Average % RSD Std. Deviation

Sample name Afatinib / Precision Afatinib / Precision Afatinib / Precision Afatinib / Precision Afatinib / Precision Afatinib / Precision

Retention time 10.548 10.549 10.532 10.557 10.552 10.544 10.547 0.082 0.009

Area 7222422 7224177 7216783 7239759 7240644 7266831 7235103 0.253 18302

Linearity: Aliquots of standard afatinib dimaleate stock solution were taken in different 10 ml volumetric flasks and diluted up to the mark with the mobile phase such that the final concentrations of afatinib dimaleate are in the range of 0.36-0.12 mg/ml. Each of these drug solutions (10 μL) was injected two times into the column, and the peak areas and retention times were recorded. Evaluation was performed with PDA detector at 258 nm and a Calibration graph was obtained by plotting peak area versus concentration of afatinib dimaleate Fig.3. The linearity Chromatograms presented in Fig.3.1- 3.5 and the values of retention time and peak area for respective concentrations were given in Table no.:3.

Fig.3.1: Leniarity for 40% (0.12mg/ml)



Fig.3.5: Leniarity for 120% (0.36mg/ml.

www.iajpr.com

Page

2104


Vol 5, Issue 05, 2015.


ISSN NO: 2231-6876

Fig 3: Standard Calibration Curve of Afatinib dimaleate. Table 3: Standard calibration values of Afatinib dimaleate. Concentration of drug (mg/mL) 0.12 0.18 0.24 0.30 0.36

Retention time 10.697 10.704 10.669 10.636 10.651

Peak Area 3016456 4403090 6094842 7261317 8806759

Interference Studies: The effect of wide range of intermediates and other additives, usually present in the pharmaceutical dosage forms for batch production, in the determination under optimum conditions were investigated. Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include impurities, degradants, matrix, etc. The commonly used reagents and intermediates present in the afatinib dimaleate tablet sample did not interfere with the elution or quantification of the method. Analysis of Afatinib dimaleate tablet: To find out the suitability of the proposed method for the assay of afatinib dimaleate in pharmaceutical dosage forms, the sample solutions from Gilotrif® tablet containing afatinib dimaleate was analyzed by the proposed method. A formulated tablet dosage form of 20mg afatinib dimaleate equivalent to 29.56 mg of the active ingredient was mixed with 30 ml of diluent in 50 ml volumetric flask. The mixture was allowed to stand for 30 minutes with intermittent sonication for complete solubility of the bulk drug, and then filtered through a 0.45 μm membrane filter, followed by addition of diluent up 50 ml to obtain a stock solution of 0.6mg/mL as the working sample solution. The results obtained from the proposed method were evaluated compared statistically. The results were found that the proposed method do not differ significantly in precision and accuracy from the statistically accepted criterion. The results are recorded in Table 4

Page

2105

Recovery Studies: Recovery studies were conducted by analyzing pharmaceutical formulation in the first instance for the active ingredient in the concentration of 80% of the working standard (contains 0.24 µg/mL); 100% of the working standard solution (contains 0.3mg/mL) and 120% of the working standard solution (contains 0.36mg/mL) by the proposed method. Each concentration was injected 3 times and the peak area was recorded. Known amounts of pure drug [25% of the working standard solution contains 250 µg/mL of Afatinib dimaleate for 50% of the working standard, 50% of the working standard solution contains 250 µg/mL of Afatinib dimaleate for 100% of the working standard, 75% of the working standard solution contains 250 µg/mL of Afatinib dimaleate for 150% of the working standard ] was then added to each 3 previously analyzed formulation and the total amount of the drug was once again determined by the proposed method (each concentration was again injected 3 times) after keeping the active ingredient concentration within the linearity limits. The Recovery chromatograms are shown in Fig. 4.1 - 4.3.

www.iajpr.com

Vol 5, Issue 05, 2015.


ISSN NO: 2231-6876

Figure 4.1: Recovery Chromatograms of Afatinib dimaleate for 80%.

Figure 4.2: Recovery Chromatograms of Afatinib dimaleate for 100%.

Figure 4.3: Recovery Chromatograms of Afatinib dimaleate for 120%. Table 4: Recovery Peak areas of Afatinib dimaleate: Peak Area 6953149 8331730 9500140

Peak Area 6946013 8346779 9506521

Avg. peak area 6951784 8373115 9500129

% Recovery 82.70 115.90 110.80 103.13

ROBUSTNESS: A method is robust if it is unaffected by small changes in operating conditions. To determine the robustness of this method, the experimental conditions were deliberately altered at three different levels and retention time and chromatographic response were evaluated. One factor at a time was changed to study the effect. Variation of the different column utilization, the mobile phase flow rate was varied by ±10%), had no significant effect on the retention time and chromatographic response of the method, indicating that the method was robust. When the chromatographic conditions were deliberately altered, system suitability results remained within acceptance limits and selectivity for individual substance was not affected which was given in table no.: 5 with chromatographic spectras fig. 5.1 – 5.6. The results of the study prove the robust nature of the method.

www.iajpr.com

2106

Peak Area 6956191 8440835 9493727

Page

Recovery Conc. in mg/mL 80% 0.24 100% 0.3 120% 0.36 Average % recovery:

Vol 5, Issue 05, 2015.


ISSN NO: 2231-6876

Table 5: Robustness values for Afatinib (Std.) and Gilotrif (Sample) compared with actual: Robustness parameters Actual Different Column Flow decrease Flow increase Different Column

Standard (Afatinib) RT Area 10.548 7234000 10.676 7193083 11.256 8138637 11.106 6520954 10.676 7193083

Sample (Gilotrif) RT Area 10.551 7189536 10.657 7255213 11.241 8185290 11.003 6561563 10.657 7255213

Fig 5.1: Chromatograms to illustrate robustness study of Afatinib dimaleate Standard solution for Different column.

Fig 5.2: Chromatograms to illustrate robustness study of Afatinib dimaleate Sample solution for Different column.

Page

2107

Fig 5.3: Chromatograms to illustrate robustness study of Afatinib dimaleate Standard solution for Decreased flow rate.

www.iajpr.com

Vol 5, Issue 05, 2015.


ISSN NO: 2231-6876

Fig 5.4: Chromatograms to illustrate robustness study of Afatinib dimaleate Sample solution for Decreased flow rate.

Fig 5.5: Chromatograms to illustrate robustness study of Afatinib dimaleate Standard solution for increased flow rate.

Fig 5.6: Chromatograms to illustrate robustness study of Afatinib dimaleate Sample solution for increased flow rate.

Page

2108

Limit of Detection [LOD] and Limit of Quantification [LOQ]: The detection limit of the method was investigated by injecting standard solutions of Afatinib dimaleate into the HPLC system. By using the signal-to-noise method the peak-to-peak noise around the analyte retention time is measured, and subsequently, the concentration of the analyte that would yield a signal equal to certain value of noise to signal ratio is estimated. A signal-to-noise ratio (S/N) of 3 is generally accepted for estimating LOD and signal-to-noise ratio of 10 is used for estimating LOQ. The LOQ can be determined by a signal-to-noise ratio of 10:1, or approximated by multiplying the LOD by 3.3. This method is commonly applied to analytical methods that exhibit baseline noise. Limit of detection (LOD) for Afatinib dimaleate is 0.06μg/mL and Limit of quantification (LOQ) for Afatinib dimaleate is 0.18μg/mL. The results of LOD and LOQ were indicating a high sensitivity of the method, which are well within the acceptable criteria of not more than 2.0. The Chromatograms illustrating the LOD along with the peak areas are shown in Fig-6&7.

www.iajpr.com

Vol 5, Issue 05, 2015.


ISSN NO: 2231-6876

Fig 6: Chromatograms to illustrate LOD having 0.02 %( 0.06μg/mL).

Fig 7: Chromatograms to illustrate LOQ having 0.06% (0.18μg/mL). Table 6: Optical & Regression Characteristics of HPLC method:

CONCLUSION There are no reports on the HPLC determination of Afatinib dimaleate in bulk and pharmaceutical formulation (tablet) in the literature prior to commencement of this work. The author has developed a sensitive, accurate and precise HPLC for the estimation of Afatinib dimaleate in bulk drug and formulation Gilotrif. From the typical chromatogram of Afatinib dimaleate as shown in fig 1.2, it was found that the retention time was 10.58 min. The contents of the mobile phase were Solvent-A is potassium di-hydrogen orthophosphate buffer system adjusted pH-3.0 with o-phosphoric acid and Solvent-B is Acetonitrile:methanol (70:30v/v) in a gradient mode as shown in table No.1.4of separation was used to resolute the Afatinib dimaleate at a flow rate of 1.0 ml/min was found to be most suitable to obtain a peak well defined and free from tailing. In the present developed HPLC method, the standard and sample preparation required less time and no tedious extraction were involved. A good linear relationship (r 2=0.998) was observed between the concentration range of 0.36-0.12 mg/mL where the optical and regression characteristics of HPLC method was given in table no.6. The assay of Afatinib dimaleate in tablet dosage form was found to be 99.38%. From the recovery studies it was found that about 103.13% on average of Afatinib dimaleate was recovered which indicates high accuracy of the method.

www.iajpr.com

2109

Results of HPLC Method 258nm 0.12-0.36mg/mL 2E+07 50343 0.998 0.03 0.15%

Page

Parameter Detection wavelength (nm) Linearity range (µg/mL) Regression Equation (y=mx + c) Slope (m) Intercept (c ) Correlation coefficient Relative Standard deviation* % error in bulk samples * Average of six determinations.

Vol 5, Issue 05, 2015.


ISSN NO: 2231-6876

www.iajpr.com

Page

REFERENCES 1. Kabbinavar FF, Hambleton J, Mass RD, Hurwitz HI, Bergsland E and Sarkar S: Combined analysis of efficacy: the addition of bevacizumab to fluorouracil/leucovorin improves survival for patients with metastatic colorectal cancer. J Clin Oncol 23: 37063712, 2005. 2. Saltz LB, Clarke S, Diaz-Rubio E, Scheithauer W, Figer A, Wong R, Koski S, Lichinitser M, Yang TS, Rivera F, Couture F, Sirzen F and Cassidy J: Bevacizumab in combination with oxaliplatin based chemotherapy as first-line therapy in metastatic colorectal cancer: a randomized phase III study. J Clin Oncol 26: 2013-2019, 2008. 3. Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, Berlin J, Baron A, Griffing S, Holmgren E, Ferrara N, Fyfe G, Rogers B, Ross R and Kabbinavar F: Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 350: 2335-2342, 2004. 4. Griesinger F, Pavlakis N, Garrido P, Dansin EE, L. Crino L, N.Thatcher N, J. Laskin J, Zhou C, Kohlhaufl MJ, Tsai C-M and group obotMSs. MO19390 (SAIL): safety and efficacy of first line bevacizumab plus chemotherapy in elderly patients with a advanced or recurrent non-squamous non-small cell lung cancer (NSCLC). World Lung Cancer Congress Proceedings, San Francisco, CA, USA, 2009. Abstract C352.356. 5. Nicholson RI, Gee JM and Harper ME: EGFR and cancer prognosis. Eur J Cancer 37(Suppl 4): S9-15, 2001. 6. Cappuzzo F: EGFR FISH versus mutation: different tests, different end-points. Lung Cancer 60: 160-165, 2008. 7. Bailey R, Kris M, Wolf M, Kay A, Averbuch S, Askaa J, Janas M, Schmidt K and Fukuoka M: Gefitinib (‘Iressa’, ZD1839) monotherapy for pretreated advanced non-small-cell lung cancer in IDEAL 1 and 2: tumor response is not clinically relevantly predictable from tumor EGFR membrane staining alone. Lung Cancer 41: S71, 2003. 8. Hoda D, Simon GR and Garrett CR: Targeting colorectal cancer with anti-epidermal growth factor receptor antibodies: focus on panitumumab. Ther Clin Risk Manag 4: 1221-1227, 2008. 9. Cunningham D, Humblet Y, Siena S, Khayat D, Bleiberg H, Santoro A, Bets D, Mueser M, Harstrick A, Verslype C, Chau I and Van Cutsem E: Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J Med 351: 337-345, 2004. 10. Jonker DJ, O’Callaghan CJ, Karapetis CS, Zalcberg JR, Tu D, Au HJ, Berry SR, Krahn M, Price T, Simes RJ, Tebbutt NC, van Hazel G, Wierzbicki R, Langer C and Moore MJ: Cetuximab for the treatment of colorectal cancer. N Engl J Med 357: 20402048, 2007. 11. Van Cutsem E, Peeters M, Siena S, Humblet Y, Hendlisz A, Neyns B, Canon JL, Van Laethem JL, Maurel J, Richardson G, Wolf M and Amado RG: Open-label phase III trial of panitumumab plus best supportive care compared with best supportive care alone in patients with chemotherapy-refractory metastatic colorectal cancer. J Clin Oncol 25: 1658-1664, 2007. 12. Van Cutsem E, Kohne CH, Hitre E, Zaluski J, Chang Chien CR, Makhson A, D’Haens G, Pinter T, Lim R, Bodoky G, Roh JK, Folprecht G, Ruff P, Stroh C, Tejpar S, Schlichting M, Nippgen J and Rougier P: Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer. N Engl J Med 360: 1408-1417, 2009. 13. Kawada I, Soejima K, Watanabe H, Nakachi I, Yasuda H, Naoki K, Kawamura M, Eguchi K, Kobayashi K and Ishizaka A: An alternative method for screening EGFR mutation using RFLP in non-small cell lung cancer patients. J Thorac Oncol 3: 10961103, 2008. 14. Schiller JH: Small cell lung cancer: defining a role for emerging platinum drugs. Oncology 63: 105-114, 2002. 15. Herbst RS, O’Neill VJ, Fehrenbacher L, Belani CP, Bonomi PD, Hart L, Melnyk O, Ramies D, Lin M and Sandler A: Phase II study of efficacy and safety of bevacizumab in combination with chemotherapy or erlotinib compared with chemotherapy alone for treatment of recurrent or refractory non small-cell lung cancer. J Clin Oncol 25: 4743-4750, 2007. 16. Herbst R, Sun Y, Eberhardt W, Germonpre P, Saijo N, Zhou C, Wang J, Li L, Kabbinavar F, Ichinose Y, Qin S, Zhang L, Biesma B, Heymach J, Langmuir P, Kennedy S, Tada H and Johnson B: Vandetanib plus docetaxel versus docetaxel as second-line treatment for patients with advanced non-small-cell lung cancer (ZODIAC): a double-blind, randomised, phase 3 trial. Lancet Oncol 11: 619-626, 2010. 17. John T, Liu G and Tsao MS: Overview of molecular testing in non-small-cell lung cancer: mutational analysis, gene copy number, protein expression and other biomarkers of EGFR for the prediction of response to tyrosine kinase inhibitors. Oncogene 28(Suppl 1): S14-23, 2009. 18. Olivier Bouche, Frederique Maindrault-Goebel, Michel Ducreux, Gerard Lledo, Thierry Andre, Peter Stopfer, Nadia Amellal, Michael Merger And Aimery De Gramont: Phase II Trial of Weekly Alternating Sequential BIBF 1120 and Afatinib for Advanced Colorectal Cancer. Anticancer Research 31: 2271-2282, 2011. 19. Medscape Reference. WebMD. Retrieved 28 January 2014.

2110

This demonstrates that the developed HPLC method is simple, linear, accurate, sensitive and reproducible. Thus, the developed method can be easily used for the routine quality control for bulk and tablet dosage form of Afatinib dimaleate within a short analysis time. It can be seen from the results presented that the proposed procedure has good precision and accuracy. Results of the analysis of pharmaceutical formulations revealed that proposed methods are suitable for their analysis with virtually no interference of the usual additives present in the pharmaceutical formulations. The above proposed method obviates the need for any preliminary treatment and the method could be of use for process development as well as quality assurance of Afatinib dimaleate in bulk drug and formulation.

Vol 5, Issue 05, 2015.


ISSN NO: 2231-6876

20. Minkovsky N, Berezov A (December 2008). "BIBW-2992, a dual receptor tyrosine kinase inhibitor for the treatment of solid tumors". Curr Opin Investig Drugs 9 (12): 1336–46. 21. Unité fonctionnelle de Pharmacocinétique et Pharmacochimie, Review of therapeutic drug monitoring of anticancer drugs part two-Targeter therapies, European Journal of Cancer Volume 50, Issue 12, August 2014, Pages 2020–2036. 22. Bersanelli M, Tiseo M, Artioli F, Lucchi L, Ardizzoni A. Gefitinib and afatinib treatment in an advanced non-small cell lung cancer (NSCLC) patient undergoing hemodialysis. anticancer Res. 2014 Jun;34(6):3185-8. 23. Peter Stopfer, Kristell Marzin, Hans Narjes, Dietmar Gansser, Mehdi Shahidi, Martina Uttereuther-Fischer, Thomas Ebner. Afatinib pharmacokinetics and metabolism after oral administration to healthy male volunteers. Cancer Chemother Pharmacol (2012) 69:1051–1061.

Page

2111

54878478451150322

www.iajpr.com